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17 iCIEF-MS


The AES patented CDTT allows a flexible and straightforward iCIEF-MS separation and characterisation of protein charge variant purely based on pI. There are many unparalleled advantages associated with CDTT iCIEF-MS


1. Minimal iCIEF-MS development is necessary. Identical iCIEF conditions can be directly transferred by selecting make up liquid and MS conditions.


2. Protein charge variants were separated in CDTT iCIEF-MS based on pI at iCIEF and measured the corresponding molecular weight at MS. Unlike other CIEF-MS or iCIEF-MS that involves chemical mobilisation, charge variant peaks are sequentially forced by hydrodynamic force from the separation capillary to the transfer capillary. While in the separation capillary, due to the high electric field and continuous migration of hydroxide ions from the cathodic and protons from the anodic side, the charge variant peaks enter the transfer capillary purely based on pI. There is no need for extra electrolyte switching after focusing, since no chemical mobilisation is involved in the mobilisation process. These greatly simplified iCIEF-MS procedures enable straightforward and robust operations.


3. Coupling CDTT iCIEF to MS works more like HPLC-MS than CE-MS. The electric circuit is completed at the separation capillary, and no current is necessary at the transfer capillary. Preferably both ends of the transfer capillary should be grounded. Therefore, there is great flexibility being able to connect the transfer capillary directly to a low flow ESI or nano ESI.


4. It is possible to conduct intact protein iCIEF-MS under denatured (at extreme pH) or native (at neutral pH) conditions with CDTT iCIEF. With direct iCIEF to low flow ESI or nano ESI, a make-up liquid at 1-20 times the mobilisation flow-rate can be applied. The composition of the make-up liquid can be any that is suitable to MS analysis. Consequently, protein charge variant iCIEF- MS can be achieved under denatured or native conditions by controlling the pH and composition of the make-up liquid.


5. CDTT iCIEF-MS permits multiple MS scans of a charge variant peak since once the charge variant peaks enters the smaller ID transfer capillary, it expands inside the transfer capillary. Although there is Tylor dispersion associate with the bulk movement, a higher purity in the centre section of the expanded peak can be expected. The significantly expanded peaks allow more MS scans for each individual peak, and the higher variant purity at the


Figure 6: iCIEF UV profile and TIC profile of the iCIEF-MS of NISTmAb. Table 2: The purity of collected charge variant peaks.


Fraction# Basic Peak 2 Basic Peak 1 Main Acidic Peak 1 Acidic Peak 2 1 2 3 4 5


90%


100% 3%


79% 29% 26%


18% 51% 40%


20% 34%


Figure 5: NISTmAb iCIEF E-gram in prep cartridge, and charge variant peak fraction confirmations.


centre section of the expanded peak also facilitates charge variant identification.


6. CDTT iCIEF-MS enables novel applications. Professor Neusus’ group demonstrated iCIEF-CZE-MS. Based on the same principle, iCIEF-nanoHPLC-MS and iCIEF-enzyme reactor transfer capillary-MS are possible.


NISTmAb is a well-studied molecule and was selected to illustrate CDTT iCIEF-MS.


The analytical iCIEF required 2M urea and 0.35% MC in the sample solution. Since MC and urea are not MS-compatible, 20% formamide was added to replace the urea and MC. The existing iCIEF method was applied to CDTT iCIEF-MS, with the make- up solution of 1% formic acid and 50% acetonitrile. The mobilisation speed was 0.05 µL/min, and the make-up solution was 5 µL/min. It can be calculated that the eluted


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